Enhanced aerobic fermentation methods for producing edible fungal mycelium blended meats and meat analogue compositions

ABSTRACT

Provided herein are shelf-stable protein food ingredients, food products comprising the shelf-stable protein food ingredients, methods of their production, and methods of their use. The shelf-stable protein food ingredients comprise cultured fungal biomass and a limited amount of water. Advantageously, the shelf-stable protein food ingredients can be stored, transported, and delivered within the food supply.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisionalapplication No. 67/733,925, entitled Enhanced Aerobic FermentationMethods for Producing Edible Fungal Mycelium Blended Meats and MeatAnalogue Compositions, and filed Sep. 20, 2018, the content of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are shelf-stable protein food ingredients, food productscomprising the shelf-stable protein food ingredients, methods of theirproduction, and methods of their use.

BACKGROUND

To keep up with global population growth projected to reach 10 billionby 2050, we must be able to produce 70% more food on Earth. The averageAmerican consumes over 200 pounds of meat per year. Meat as a proteinsource is becoming increasingly costly to humanity due to associatedhabitat conversion, water, and resource use. As environmental resourcesbecome more scarce, we cannot afford to invest valuable nutrientresources into inefficient food production processes such as farming ofanimals. The tasks of reducing meat consumption along with creatingsustainable alternatives will be required to meet the protein demand ofthe future. On average, 5% of the calories from animal feed make it intothe meat consumed by humans. These vital nutrients are only becomingmore precious a commodity. The environmental budget of the planet willnot allow humans to continue under such tremendous inefficiency.

Technical Problem

The alternative protein industry is booming. The current landscape ofplant protein ingredients is incredibly limited. Almost every product onthe market relies on either wheat, soy, or pea proteins. There are tworealms of texturization that companies use to produce their meatanalogues—wet texturization and dry texturization. Virtually alltexturized plant protein sold today as an ingredient is as a drytexturized protein. These ingredients are limited to the versatility oftwin-screw extrusion systems, and the functionality of the threeaforementioned plant protein inputs. The ingredients being produced arevery similar to each other, and the two most widely used proteins (wheatand soy) are both in the list of the eight most common allergens.Texturized pea protein is still very expensive. In order to move theindustry forward, we need alternative ingredients for the plant-basedprotein world.

Cutting the amount of animal-derived meat needed to produce a meatproduct is a concept being investigated by many key players in the meatindustry. Certain ingredients in existing blended meats lack theprotein, texture, and filamentous nature of muscle and fat in meat.These ingredients, described as meat extenders, typically only consistof single ingredient, e.g. flours, powders, or crumbs, lacking nutritionand enhancement of the meat.

The food and beverage industries produce high volume food grade wastestreams containing quality starches, sugars, nitrogen, fats, vitamins,minerals, and other nutrients. This material often goes to feed animalsat an inefficient conversion rate or is treated as a complete waste andsent to landfills or treatment facilities. Leftover material fromprocessing, extraction, and cultivation are available on a consistentbasis. These are valuable food grade nutrients that we cannot afford tolose.

Commercial plant agriculture is dependent on environmental parametersbeyond the control of man. Weather, climate, wind, pathogens, blight,disasters, and more can unpredictably wipe out entire areas of foodproduction. Controlled agriculture is essential; however, it is limitedto indoor plant farming reliant on artificial lighting powered primarilyby the electric grid and in effect, coal and natural gas.

Significant resources are being directed towards sustainable methods ofproducing cultured meats and meat analogues. Meat alternatives in themarketplace are mostly derived from plant biomass. Plant based analoguesand meat fillers commonly require significant manipulation and additivesto mimic the texture, nutritional profile, and properties of meat.

Current meat analogues rely almost entirely on texturized vegetableproteins (TVP). These ingredient supplies are reliant on costlytexturization processes, limited manufacturing capacity, environmentalimpacts on cultivation, supply of raw material, and often timesprocessing aids. These ingredients have functional limitations as theycan only achieve certain textures and qualities. The base plant biomassof most TVP is wheat and soy, two of the eight common allergens andtherefore not preferred as ingredients. Some texturized pea exists inthe marketplace, however, it is limited in use by its high cost andconstricted supply.

Solution

Provided herein are shelf-stable protein food ingredients that areversatile in production methods, functionality, and form. Theingredients can be completely allergen free and potentially cheaper thantexturized pea protein. The shelf-stable protein food ingredients canhave substantial impacts in the types of alternative protein foodingredients and products in the marketplace.

In one aspect, provided herein are fermented shelf-stable protein foodingredients that can be dry, stored, and readily integrated into thefood ingredient supply chain. These ingredients are designed to bemeat-like, versatile, and cost-effective to produce. They can be readilysubstituted for plant based protein ingredients and can be produced fora lower cost in many embodiments.

In one aspect, provided herein are fermentation processes that harnesswaste nutrient streams from crop and food processing. Harnessing thesestreams has a multitude of benefits for society, and our planet as awhole. By utilizing these nutrient streams and converting them into highquality food ingredients and products, we are garnering a qualityprotein source without requiring significant land and carbon impacts onthe planet compared to meat or even plants.

Fungi are decomposers. They complete degradation of complex organicmolecules like lignin and cellulose in nature. By excreting digestiveenzymes and acids, fungi are able to convert more complex carbohydratesinto simple fermentable sugars. Their incredible efficiency atdecomposing and consuming low value waste streams can be utilized toproduce high value, quality food for the masses with low-cost,inexpensive inputs.

In one aspect, provided herein are shelf-stable protein food ingredientsthat create a means of reducing and replacing animal meat products.These shelf-stable protein food ingredients can provide all essentialamino acids (significant amounts of protein), fiber, quality fats, andmost essential micronutrients and vitamins. In certain embodiments, theproduction process is efficient and environmentally sustainable. Inanother aspect, processes for producing these foods are disclosedherein.

The shelf-stable protein food ingredients described herein can beproduced in a refined, controlled, environment that mitigates most ofthe problems associated with traditional agriculture. The shelf-stableprotein food ingredients can comprise fungi cultivated in optimizedfermentation systems with low physical, financial, and environmentalfootprints. These systems are more typically utilized in the productionof penicillin, enzymes, and acids but can be adapted for use in theproduction of food ingredients.

Filamentous fungal mycelium has been consumed for centuries in the formsof tempeh, oncom, koji, and other foods, some of which are mentionedbelow. These products consist of filamentous fungi grown on solidsubstrates. Soy, rice, and other solid substrates are nutritious;however, these solid substrates remain in the end product and dilute thepotential end protein/nutrient content. These ingredients also holddietary value on their own and do not require the fungi to becomevaluable to the human diet. They may be enhanced by the fungi; however,the fungal cells play a minor role in the end product.

In certain embodiments, the food ingredients and food products providedherein comprise fungal species established in human consumption and havestood the test of time. Aspergillus oryzae is the species used to makeKoji (a fermented rice used in the production of sake, miso, and soysauce). Rhizopus oryzae is a species used in fermenting tempeh.Cordyceps militaris and Cordyceps sinensis are edible fungi used intraditional Chinese Medicine. Tuber magnatum, better known as the BlackTruffle, is an edible fungus known for its array of aromatic compoundsthat are responsible for its sought after flavor. Fusarium has beenutilized as a protein source. Penicillium is used in cheese making.Neurospora intermedia and Nerospora sitophila are used to ferment oncom(a fermented tempeh-like soy, peanut, or legume food very commonlyconsumed on the mainland of Indonesia (Java) for centuries).

Filamentous fungi are known for their rapid cell replication, aggressivedigestion, colonization timing, adaptability, and ease of propagation.This makes them well suited for scaled food production.

Food Ingredients

Filamentous fungal mycelium maintains a texture similar to ground meatwith minimal manipulation. Filamentous fungi mycelium described hereincomprises groups of connected cells fused end to end in filaments calledhyphae. These hyphae typically range from 2-16 microns in diameter andcan be centimeters long. These hyphae are typically one single cellthick. These morphologies give the hyphae naturally occurring textureproperties similar to muscle fiber as a result of the bundling of thehyphae and the substantial moisture retention capacity of the mycelium.This makes mycelium a perfect candidate for food ingredients and foodproducts.

In certain embodiments, the fungal mycelium is processed into ashelf-stable protein food ingredient that can be hydrated and used onits own or, advantageously, in a variety of food products including butnot limited to meat extenders, meat analogues, cultured meat cellscaffoldings, and other food products requiring textured proteins. Asused herein, these “textured cultured proteins” (hereinafter “TCP”) aredry, shelf stable, and easily used as a replacement for lower qualitymore expensive texturized vegetable proteins (hereinafter “TVP”).

In certain embodiments, filamentous fungal mycelium described hereincomprises significant concentrations of nutrients. In certainembodiments, crude protein accounts for up to 60% of the untreateddesiccated biomass. Most species contain all of the essential aminoacids of the human diet. Many species contain all of the necessary Bvitamins when un-supplemented (except B12). The mycelium contains manydietary minerals needed in the human diet including but not limited tozinc, iron, manganese, magnesium, potassium, selenium and calcium. Allof these minerals except for zinc have been shown to be morebioavailable to the body when sourced from mushrooms and fungi comparedto meat and plant sources. The mycelium is naturally high in fiber.

In certain embodiments, the fat composition of the mycelium describedherein comprises or consists of mostly mono- and polyunsaturated fatsand is very low in saturated fats. The biomass also may contain omega-6,linoleic acid, and omega-3, linolenic acid.

In certain embodiments, the dry shelf-stable ingredients describedherein comprise significant fiber. This substantial concentration ofdietary fiber in the TCP is beneficial when consuming TCP as a meatalternative, as well as being beneficial in meat/TCP blends. The presentfiber increases the digestibility and bioavailability of the nutrientsin the meats consumed in the blend.

It is therefore an aspect described herein to produce a food ingredientusing fungi, many species of which are already accepted in theirrecognition of safety in the diet of humans and significantly higher inprotein and fungal cells than classic fermented foods that contain someof these species.

Phosphates are commonly used in meat products to increase moistureretention by creating space between proteins. Provided herein arefilamentous mycelium that can retain over 80% water. At levels of 60-85%water content the dry shelf-stable ingredient of the present inventionmixes well with meats and acts as a tackifying agent helping to bind themeat while holding moisture in the meat mixture. The filamentous natureof the mycelium maintains natural space between proteins. This moisturehelps the meat retain its volatile aromatics effectively preventingflavor loss. This may have implications is shelf life extension of somemeat products. Upon dehydration, the mycelium described herein canremain shelf stable and retain a similar water content to the freshmaterial when re-hydrated.

Process

By using fermentation to produce these high protein texturedingredients, carbohydrates, other nutrients, and oxygen are convertedinto protein. Such methods can use less space, resources, and time thanthose of conventional conversions in animal agriculture.

Filamentous fungi fermentation can be carried out with waste streams asprimary sources of nutrients, displacing the need to introduce so manypurified nutrients into the growing medium.

Beet pulp, potato peel and processing water, processed grains, processfruits, rice polishings, and much more are abundant and available andcan be used for sourcing of components in fungal fermentation substrate.Filamentous fungi effectively convert sugars and starches from thesesources into biomass at a high efficiency. Some industrial fermentationoperations, such as Cargill's lactic acid plant in Blair, Nebr., USA,employ these concepts. Cargill uses beet pulp from refining sugar at theBlair plant for their primary carbohydrate source for the production oflactic acid.

In some embodiments more traditional fermentation substrates are usedfor producing the TCP described herein.

Food Products

Converting diet to meat alternatives has a place in the changing diet ofhumans; however, meat consumption is unlikely to be entirely replaced.The concept of extended meats has existed in the art for years. Bysimply extending the meat in processed meat products, one cansignificantly reduce overall meat production and demand. Currentextension agents provide only some of the needed properties to have anindistinguishable profile in the blended meat. Mushrooms likeportabellas are sometimes used to dilute meat and retain moisture, butonly provide some of the desired properties of meat extenders whilelacking others.

In certain embodiments the food ingredients described herein provide analternative to texturized vegetable protein (TVP), the core ingredientin most meat analogues on the market.

It is therefore an aspect described herein to utilize the foodingredients provided herein for applications including but not limitedto meat extension products, meat analogue products, baked good products,food products requiring binding agents, food products requiring gels,food products needing protein, food products needing fiber, and otherfood products.

SUMMARY

In one aspect, provided herein are shelf-stable protein foodingredients. The shelf-stable protein food ingredients comprise culturedfungal biomass and a limited amount of water. The fungal biomass andother ingredients are described in detail herein. The shelf-stable foodingredients comprise particles of sizes and forms with propertiesdescribed herein. Advantageously, the shelf-stable protein foodingredients can be stored, transported, and delivered within the foodsupply. They can be sold or consumed as is, or, preferably, they can becombined with other food ingredients to provide food ingredientcompositions and food products.

In another aspect, provided herein are food products. The food productscomprise one or more of the shelf-stable protein food ingredients andone or more additional food ingredients. The additional food ingredientscan be meat proteins, plant proteins, combinations thereof, or anyadditional food ingredient deemed useful by the practitioner of skill inthe art. The food products can be consumed by animals, for instancemammals. In certain embodiments, the food products are for petconsumption. In certain embodiments, the food products are for humanconsumption.

In another aspect, provided herein are methods for producing theshelf-stable protein food ingredients. In certain embodiments, themethods comprise the steps of culturing fungal biomass in a growthmedium, harvesting the fungal biomass, optionally processing the fungalbiomass, optionally sizing the fungal biomass to form particles, anddrying the particles to form the food ingredients.

In certain embodiments, provided herein are methods of producing ameat-textured high protein ingredient from filamentous fungal myceliumproduced with plant biomass hydrolysate as the primary growth media. Themethods comprise the steps of generating plant biomasshydrolysate/extract; enhancing the hydrolysate with supplements toenhance yields, nutritional profile, and morphology; sterilizing saidsubstrate; inoculating substrate with filamentous fungi; propagating thefilamentous fungus in optimized aerobic fermentation conditions;harvesting pure fungal mycelium, de-watering, shaping, sizing, dryingand pasteurizing; integrating dried and shaped ingredient into blendedmeat extension ingredients; hydrating and blending hydrated ingredientin ground meats; and utilizing aforementioned ingredients in blendedplant and mushroom products.

In certain embodiments, provided herein are methods of producing ashelf-stable protein ingredient from filamentous fungal mycelium withsubstrates based on glucose, sucrose, sugars, starches, and/or biotin aswell as salt forms of nitrogen, phosphorous, potassium and othernecessary elements. The methods comprise mixing said substrate;sterilizing said substrate; inoculating substrate with filamentousfungi; propagating the filamentous fungi in optimized aerobicfermentation conditions; harvesting pure fungal mycelium, de-watering,shaping, sizing, drying and pasteurizing; integrating dried and shapedingredient into blended meat extension ingredients; hydrating andblending hydrated ingredient in ground meats; and utilizingaforementioned ingredients in blended plant and mushroom products.

In certain embodiments, provided herein are methods for fermentingfilamentous fungi with carbohydrate rich and other raw plant biomass toproduce meat-textured ingredients. These methods may entail introductionof plant flour, granules, grains, legumes, or combinations thereof orother plant biomass into the fermentation liquid described herein.

In certain embodiments, provided herein are methods for converting theharvested fungal mycelium into a meat like particulate dry ingredient.In some embodiments, the ingredient is combined with other ingredientsfor functional enhancement; in some embodiments, the ingredient is usedas a stand-alone ingredient; in some embodiments, the ingredient ishydrated with water and integrated into meats; in some embodiments, theingredient is hydrated and integrated into meat analogues including butnot limited to burgers, sausages, patties, nuggets, and more.

In another aspect, provided herein are food compositions comprising ashelf-stable protein food ingredient and one or more meats. Inparticular embodiments, the food compositions comprise the shelf-stableprotein food ingredient in an amount of at least 5% w/w and at least onemeat in an amount of at least 10% w/w. Shelf-stable protein foodingredients and useful meats are described in detail in the sectionsbelow, along with methods of preparing the food compositions.

In an embodiment, provided herein are methods for processing fungalmycelium described herein into a pasteurized biomass that can be blendedinto meat to make blended meat products described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an overview of an embodiment of the fermentation processused to produce the biomass described herein.

FIG. 2 provides an overview of some methods for processing of the fungalbiomass slurry into the textured cultured protein (TCP) describedherein.

FIG. 3 provides an overview of one embodiment of the fungal biomassfermentation described herein.

FIG. 4 provides an overview of optional applications of some of theingredients described herein.

FIGS. 5A and 5B provide photographs of exemplary shelf-stable proteinfood ingredients provided herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Methods of Production

In particular embodiments, provided herein are methods for producingshelf-stable protein food ingredients. In certain embodiments, themethods comprise the steps of culturing filamentous fungi in growthmedium; harvesting filamentous fungal biomass; optionally, processingthe harvested filamentous fungal biomass; optionally, sizing the biomassto form particles; and drying the particles to form the food ingredient.In certain embodiments, the methods comprise the steps of culturingfilamentous fungi in growth medium; optionally supplementing the growthmedium to form a mixed fungal biomass slurry; harvesting filamentousfungal biomass; optionally, processing the harvested filamentous fungalbiomass; optionally supplementing the processed biomass to form acomplex ingredient; optionally sizing the biomass to form pieces; anddrying the pieces to form the food ingredient.

In the culturing step, the filamentous fungus can be cultured accordingto standard techniques. The culturing typically comprises growing thefilamentous fungus in a growth medium. In certain embodiments, thegrowth medium comprises hydrolyzed plant biomass byproducts andwastewater streams of the food and beverage industries as well aslow-cost plant ingredients and other substrates.

In certain embodiments, the culture is batch culture, fed-batch culture,or continuous culture. The growth medium includes the ingredientsdescribed below. Additional additives can be provided according to thejudgment of the practitioner in the art. Culture conditions are withinthe skill of those in the art including culture volume, temperature,agitation, oxygen levels, nitrogen levels, carbon dioxide levels, andany other condition apparent to those of skill.

In certain embodiments, pure oxygen is used in the aeration of thefermentation. Injecting O₂ into the downcomer of the reactor can allowmaximum interaction with the gas bubbles and substrate while maximizingdiffusion of the O₂. Pressure swing adsorption (PSA) oxygenconcentration is sometimes used to continuously inject O₂.

The fungal fermentation can operate with a wide pH range. In certainembodiments the pH is between about pH 2.4 and about pH 8.5, betweenabout pH 3 and about pH 7.5, between about pH 3 and about pH 6.5,between about pH 3 and about pH 5.5, or between about pH 3.5 and aboutpH 4.5.

The cultured filamentous fungi can be harvested according to anystandard technique. The methods include any harvesting technique deemeduseful to the practitioner in the art. Useful techniques includecentrifugation, pressing, screening, and filtration. In certainembodiments, the filamentous fungi are de-watered and separated bycentrifugation. In certain embodiments, the filamentous fungi arede-watered and separated via screw press. In certain embodiments fungiare de-watered and separated via de-watering vibratory screen. Incertain embodiments fungi are de-watered via a fluidized bed dryer. Incertain embodiments, the filamentous fungi are washed to remove excessgrowth medium. In certain embodiments, the filamentous fungi are notwashed.

In certain embodiments, the harvested filamentous fungal biomass isprocessed for further use. The processing technique can be anyprocessing technique apparent to the practitioner of skill.

In certain embodiments, hydrolysis and starch conversion process ofplant material is accelerated by using a protease, β-glucanase,xylanase, α-amylase and/or other enzymes during the hydrolysis step ofgenerating the substrate of the aerobic fermentation.

In certain embodiments, an alkali or acid treatment is used in thehydrolysis of the plant material used as substrate.

In certain embodiments, the filamentous fungi are sized according to therequirements of the ingredients described herein. Biomass released fromde-watering processes can be cake-like as it is similar to fruit, paper,or other pulp upon de-watering. This cake can be broken apart, shredded,chopped, sliced, diced, sieved, and further reduced to desired sizeusing conventional sizing equipment before or after de-hydration. Thebiomass can be molded, pressed, rolled, extruded, compacted, ormanipulated in other ways known to a person with skill in the art ofsizing food materials. This can happen before de-watering, during,de-watering, after de-watering, before de-hydration, duringde-hydration, or after de-hydration. In certain embodiments, the biomassis sized to yield particles of sizes described below.

In certain embodiments, the biomass slurry comprises highly dispersedfilamentous cell structures. Upon removing water using gravity,pressure, compaction, vacuum suction, centrifugation, or other methodsknown to those skilled in the art of de-watering, filamentous cellularstrands interlock with each other to form cohesive filamentous mats thatmaintain consistency and cohesion. A belt press can be particularlyuseful in producing large meaty slabs of dense cohesive biomass. Thesedense mats can be sliced, diced, chopped, molded, folded, extruded, orotherwise manipulated in a way known to a person with skill in the artto form slices, chunks, shreds, nuggets, or other particles and pieces.

In certain embodiments the biomass described herein is extruded usinghigh temperature twin screw extrusion or other extrusion technology toform a material with a more conventional texture that is similar to TVP.

In certain embodiments the biomass of the fermentation process isde-watered to remove moisture from the biomass. The material isoptionally pasteurized in the substrate by using steam to heat thebiomass slurry to pasteurization temperatures (75-85° C.); the slurry isoptionally released into a vibratory screen to de-water the materialdown to 75-95% water content; the biomass is then optionally pressedwith a belt press or screw press or centrifuged to further reduce thewater content; the material is then optionally shredded, sized,compacted, molded, otherwise formed, or combinations thereof; thematerial is then optionally added to a fluidized bed dryer for fulldehydration. In particular embodiments, the material is de-watered toyield a water content for the particles as described below.

In certain embodiments the biomass slurry containing 1-8% biomass isreleased into a belt press system. The material is simultaneouslydrained and pressed bringing the material down to 60-85% water content.The machine is adjusted to release a cake/slab at a thickness of 1inch/2.54 cm. The 1 inch slab is continuously conveyed into a spindleand tine mechanical shredder. The shredder releases granular particlesin the size range of about 1 mm-about 20 mm. Particles are continuouslysieved using 2 mm and 12 mm sieves. Particles released through the 2 mmsieve are saved and de-hydrated separately or re-introduced to theinitial slurry. Particles released through the 12 mm sieve but notthrough the 2 mm sieve are fed directly into a fluidized bed dryer fordehydration and optionally pasteurization. Particles larger than 12 mmare optionally conveyed back through the shredder for further sizereduction. The dehydrated particles between about 2 and about 12 mm areready for use as a bare ingredient or to be further processed intoingredients described herein. As used herein, the term “about” indicatesa reasonable range above and below a unit value, for instance +/−10% or+/1 unit, e.g. mm.

In certain embodiments the biomass slurry is de-watered down to 50-75%water content with methods known to a person with skill in the art ofde-watering microbial biomass. The lower moisture biomass at 50-75%water content is then fed through a dough chopping machine. Material isfed through a ¼ inch die and chopped into small particles intermittentlyby a rotating shear at the end of the die. This results in chunks of aconsistent size of about (⅛ inch to ¼ inch) by (⅛ inch to ¼ inch) by (⅛inch to ¼ inch).

In certain embodiments plant or mushroom materials are added to thebiomass prior to de-hydration. These materials can be added to thefungal biomass slurry, the fungal biomass with a water content of60-85%, the fungal biomass of a water content of 60-75%, the fungalbiomass of a water content of 50-75%, the fungal biomass of a watercontent of 50-65%, or fungal biomass with other water content. Thesematerials may be blended with the fungal biomass described herein andthen further de-watered, de-hydrated, or processed into the driedtextured ingredients described herein.

In another embodiment supernatant of the fermentation process containinghigh concentrations of digestive enzymes such as alpha amylase, betaamylase, lipase, and others is processed into side streams. Theseenzymes can be extracted, purified, and sold or used in pretreatment ofhydrolyzed starch rich substrates or in other applications. Theseenzymes from the supernatant can be integrated into the meat analoguesand blends described herein to promote more effective digestion.

Growth Medium

The growth medium can be any growth medium deemed suitable. Inparticular embodiments, the growth medium comprises plant biomass. Incertain embodiments, the plant biomass is a wastewater stream. The plantbiomass co-products, ingredients, and wastewater streams can be anymaterial stream deemed suitable to the practitioner of skill in the art.In particular embodiments, the plant biomass is from a low-cost source.In certain embodiments, the plant biomass is a waste stream or aco-product from another food, agriculture, or plant processing process.In such embodiments, the methods provide a second or renewable use ofco-products that are conventionally used as farm animal feed, soilenhancement, or discarded entirely. In certain embodiments, the materialis selected from ethanol thin stillage, ethanol co-products, ricemilling co-products, rice polishings, rice bran, rice process wastewater, rice brokens, brewing and distilling spent grains, spent sakerice, spent soy sauce soy, beet pulp, coffee chaff, molasses, sugarrefinery waste water, grape pulp, soda production waste water, sugarcanebagasse, sorghum bagasse, or combinations thereof. In preferredembodiments the nutrient streams are potato processing waste-water,ethanol corn stillage,

In certain embodiments, the methods comprise fermenting plantingredients in submerged culture to create cultured plant ingredientsthat contain a significant concentration of fungal biomass.

In some embodiments, the growth medium comprises one or more plantsubstrates selected from pea fiber, other plant fibers, gum arabic,natural flavors, texturized pea protein, texturized wheat protein,texturized soy protein, soy protein, wheat starch, wheat protein, peaprotein, spices, safflower oil, sunflower oil, olive oil, other oils,oat bran, oat flour, legumes, beans, lentils, lentil powder, beanpowder, pea powder, yeast extract, nutritional yeast (immobilized driedyeast), molasses, honey, cane sugar, mushroom powder, white buttonmushroom powder, shiitake mushroom powder, chickpeas, bamboo fiber,cellulose, isolated oat product, isolated pea product, pea protein, riceprotein, fermented rice extract, corn starch, potato starch, kombuextract, algae, potato protein, albumin, pectin, silicone dioxide, foodstarch, mixed tocopherols (vitamin E), coconut oil, sunflower oil,safflower oil, rapeseed oil, canola oil, dextrose, vegetable glycerin,dried yeast, citrus extract, citrus fiber, beet pulp, beet juice, beetjuice extract, turmeric, mushroom extract, shiitake mushroom stems,shiitake mushrooms, white button mushrooms, tofu, soy fiber, soyhydrolysate, yeast extract, seaweed, malted barley, malt extract, yeastextract, whole cell yeast, lentils, black beans, pinto beans, beans,legumes, and any combination thereof. In preferred embodiments, theplant biomass is potato or corn stillage.

In certain embodiments, the growth medium is supplemented with one ormore additive components. The additive components might facilitategrowth of the filamentous fungi, they might add nutrients to theresulting food product, or they might do both. In certain embodiments,the additive components comprise one or more carbohydrates (simpleand/or complex), nitrogen, vitamins, minerals, fats, proteins, or acombination thereof.

In certain embodiments, the additive components comprise one or moreoils. In certain embodiments, the one or more oils are selected from thegroup consisting of grapeseed oil, safflower oil, sunflower oil, oliveoil, coconut oil, flaxseed oil, avocado oil, soybean oil, palm oil,canola oil, and combinations thereof.

In certain embodiments, the one or more additives comprise one or moresalts. In certain embodiments, the one or more salts consist of elementsselected from the group consisting of C, Zn, Co, Mg, K, Fe, Cu, Na, Mo,S, N, P, Ca, Cl, and combinations thereof.

In certain embodiments the salts comprise one or more of the followingsalts: ammonium nitrate, mono-potassium phosphate, di-potassiumphosphate, di-ammonium phosphate, ammonium phosphate, potassium nitrate,magnesium sulfate heptahydrate, calcium chloride dehydrate, zinc sulfateheptahydrate, iron sulfate hexahydrate, copper sulfate pentahydrate,manganese sulfate, and combinations thereof.

In certain embodiments, the growth medium comprises one or morecarbohydrates. In certain embodiments the one or more carbohydrates areselected from glucose, sucrose, starch, maltose, and any combinationthereof.

In certain embodiments, the growth medium comprises plant oils. Theplant oils can significantly increase yields, fermentation efficiency,and fat content of the end material. Non-limiting examples of plant oilscan include almond oil, avocado seed oil, cocoa butter, coconut oil,corn oil, cottonseed oil, flax seed oil, grapeseed oil, hemp oil, oliveoil, palm kernel oil, peanut oil, pumpkin seed oil, rice bran oil,safflower seed oil, sesame seed oil, sunflower seed oil, soybean oil, orwalnut oil.

In certain embodiments, the growth medium comprises vitamins. Usefulvitamins include but are not limited to vitamin A, B1, B2, B3, B5, B6,B7 (vitamin H, or Biotin), B9, B12, C, E, D, and K, for the purpose ofintegrating the vitamin into the end food product via adsorption andcellular integration in the fermentation. In an embodiment, vitamin B12is added to the fermentation.

In certain embodiments, vitamin B12 is added to the substrate. Thevitamin is accumulated by the fungal cells. It is the only B vitaminessential to the human diet that the fungus does not produce on its own.

In certain embodiments the plant-derived biomass or material is added tothe growth medium without hydrolysis.

In certain embodiments, the growth medium comprises spent malted barley.The spent malted barley can be used as a plant biomass for hydrolysis,filtration and integration into the substrate as a primary source ofcarbohydrates, fats, proteins, and micronutrients.

In certain embodiments, the growth medium comprises potato peel. Thepotato peel can be used as a plant biomass for the hydrolysis,filtration and integration into the substrate as a primary source ofcarbohydrates, fats, and micronutrients.

In certain embodiments, the growth medium comprises potato processingwastewater. The potato processing wastewater can be used as a primarynutrient source for the fermentation of the fungi. Blanching, starchextraction and other processing methods used for potato processingproduce large volumes of nutritionally consistent waste water that ishighly effective as a fermentation feedstock.

In certain embodiments waste-water from potato blanching is used assubstrate promptly after blanching. The high temperature of theblanching process pasteurizes the substrate, effectively reducingtreatment costs in addition to substrate costs. In some embodimentsother blanche water and other clean potato processing streams arediverted away from field leaching, conventional treatment methods, andother disposal methods towards the platform described herein.

In certain embodiments, the growth medium comprises beet pulp. The beetpulp can be used as a plant biomass for the hydrolysis, filtration andintegration into the substrate as a primary source of carbohydrates,micronutrients, and nitrogen.

In certain embodiments, the growth medium comprises thin stillage (aco-product of biofuel production with corn). The thin stillage can beused as a complex nutrient source of carbon, nitrogen, micronutrients,and fats.

In certain embodiments, the growth medium comprises yeast extract. Theyeast extract can be used as a source of nitrogen and micronutrients inthe fermentation media.

In certain embodiments, the growth medium comprises filtered beet pulpextract. The filtered beet pulp extract can be used for the primarycarbon source.

In certain embodiments, the growth medium comprises potato blanch andprocessing wastewater. The potato blanch and processing wastewater canbe used for the primary nutrient source.

In certain embodiments, the growth medium comprises rice polishings.Rice polishings can be left over from polishing and/or milling rice.They can be used as a plant biomass and added to water, sterilized, andintegrated into the reactor as a primary source of carbohydrates,nitrogen, potassium, and other nutrients.

In certain embodiments, the growth medium comprises common carbohydratesources such as sucrose, glucose, and molasses. They can be used withsupplementation for the substrate. These ingredients can be blended,sterilized, and integrated into the reactor with filamentous fungidescribed herein. The produced biomass can be processed into theversatile dried ingredients described herein.

Filamentous Fungi

The filamentous fungi can be any filamentous fungi deemed suitable tothe person of skill in the art.

In certain embodiments, at least one fungus is from the kingdom ofFungi.

In certain embodiments, at least one fungus is from the phylumBasidiomycota, Ascomycota, Glomeromycota, Mucoromycota, orZoopagomycota.

In certain embodiments, at least one fungus is from the divisionagaricomycotina, ustilagomycotina, pezizomycotina, saccharomycotina,taphrinomycetes, diversisporalis, archaeosporales, paraglomerales,endogonales, mucorales, mortieralles, entomophthoromycotina,asellariales, kickxellales, dimargaritales, harpellales,zoopagomycotina, or combinations thereof.

In certain embodiments, at least one fungus is from the classtremellomycetes, dacrymycetes, agaricomycetes, exobasisiomycetes,ustilaginomycetes, malasseziomycetes, moniliellomycetes,arthoniomycetes, coniocybomycetes, dothideomycetes, eurotiomyctes,geoglossomycetes, laboulbeniomycetes, lecanoromycetes, leotiomycetes,lichinomycetes, orbiliomycetes, pezizomycetes, sordariomycetes,xylonomycetes, or combinations thereof.

In certain embodiments, at least one fungus is from the orderfilobasidiales, agaricales, amylocorticiales, atheliales, boletales,jaapiales, lepidostromatales, geastrales, gomphales, hysterangiales,phallales, auriculariales, cantherellales, corticiales, gleophylalles,hymenochaetales, polyporales, russulales, sebacinales, stereopsidales,thelephorales, trechisporales, ceraceosorales, doassansiales,entyomatales, exobasidiales, georgefischeriales, microstromatales,tilletiales, urocystales, ustilaginales, malassezioales, moniliellales,saccharomycetales, coronophorales, glomeralles, Hypocreales,melanosporales, microascales, boliniales, calosphaeriales,chaetospheriales, coniochaetales, diasporthales, magnaporthales,ophiostomatales, sordariales, xylariales, koralionastetales,lulworthiales, meliolales, phylachoralles, trichosphariales, eurotiales,chaetothyriales, pyrenulales, verrucariales, onygenales, mortierellales,mucorales, endogonales, or combinations thereof.

In certain embodiments, at least one fungus is from the familyFilobasidium, Dacromycetaceae, Agaricaceae, Amanitaceae, Bolbitiaceae,Broomeiceae, Chromocyphellaceae, Clavariaceae, Cortinariaceae,Cyphellaceae, Enolomataceae, Fistulinaceae, Himigasteraceae,Hydnangiaceae, Hygrophoraceae, Inocybaceae, Limnoperdacea,Lyophyllaceae, Marasmiaceae, Mycenacea, Niaceae, Pellorinaceae,Physalacriaceae, Pleurotacea, Pluteaceae, Porotheleaceae,Psathyrellaceae, Pterulacea, Schizophyllaceae, Stephanosporaceae,Strophariaceae, Tricholomataceae, Typhulaceae, Boletaceae,Boletinellaceae, Coniophoraceae, Diplocystaceae, Gasterellaceae,Gastrosporiaceae, Gomphidiaceae, Gyroporaceae, Hygrophoropsidaceae,Paxillaceae, Protogastraceae, Rhizopogonaceae, Sclerodermataceae,Serpulaceae, Suillaceae, Tapinellaceae, Hymenochaetaceae,Repetobasidiaceae, Schizoporaceae, Cystostereaceae, Fomitopsidaceae,Fragiporiaceae, Ganodermataceae, Gelatoporaceae, Meripilaceae,Merulaciaea, Phenerochaetaceae, Polyporaceae, Sparassidaceae,Steccherinaceae, Xenasmataceae, Albatrellaceae, Amylostereaceae,Auriscalpaceae, Bondarzewiaceae, Echinodontiaceae, Hericiaceae,Hybogasteraceae, Lachnocladiaceae, Peniphoraceae, Russulaceae,Gloeocyctidiellacceae, Stereaceae, Ustilaginomycetes,Saccharomycetaceae, Saccharomycodaceae, Saccharomycopsidaceae,Chaetomiaceae, Lasiosphaeriaceae, Sordariaceae, or combinations thereof.

In certain embodiments, at least one fungus is from the genusNeurospora, Aspergillus, Trichoderma, Pleurotus, Ganoderma, Inonotus,Cordyceps, Ustilago, Rhizopus, Tuber, Fusarium, Pennicillium, Xylaria,Trametes, or combinations thereof.

In certain embodiments, at least one fungus is Aspergillus oryzae,Rhizopus oryzae, Fusarium graminareum, Cordyceps militaris, Cordycepssinensis, Tuber melanosporum, Tuber magnatum, Pennicillium camemberti,Neurospora intermedia, Neurospora sitophila, Xylaria hypoxion, or acombination thereof.

Exemplary Methods

In this section, illustrative methods of production are provided. Theyare intended to exemplify but not limit the methods described above.

In certain embodiments, plant material is soaked in water at 45° C. with0.75 g/kg alpha-amylase, 0.25 g/kg beta-amylase, 0.5 g/kgbeta-glucanase, 0.3 g/kg protease, and 0.3 g/kg xylanase (grams ofpurified enzyme to kilograms of desiccated substrate). The liquidtemperature is increased to 78° C. over the course of 30-180 minutes toactivate the enzymes and their hydrolyzing functions. Once the mixturereaches 78° C., where the enzymes are de-activated, the mixture israpidly brought to 100° C. and maintained there for 10-120 minutes tocomplete the hydrolysis. The quantities of enzymes, their weights,temperatures, interaction times and their ratios can change based on theplant material being hydrolyzed or to optimize the effect of the enzyme.

In certain embodiments the solid plant-based ingredients are blendedinto room temperature water. The slurry is pumped through a continuoussteam sterilizer and injected into the fermentation reactor describedherein at the desired flow rate for the method of fermentation beingrun. In some embodiments the slurry is pumped at the maximum rate ofsterilization to fill a sterile fermenter for the primary initiation ofa batch style fermentation. In some embodiments the slurry is injectedin pulses aligning with the extraction of biomass slurry describedherein. In some embodiments the slurries are injected continuously.

In some embodiments the solid plant-based ingredients are sterilizedwith standard techniques, known to someone with skill in the art ofsterile processes, and introduced to the fermentations of the presentinventions.

In some embodiments starch rich processing wastewater from potatoblanching, steaming, and/or general processing used as the primarysubstrate of the fermentation. In some embodiments it is steamsterilized in a continuous media sterilizer. The material may or may notbe pre-heated from the potato processing providing energetic efficiencyadvantages. This material may constitute 100% of the growth mediadescribed herein. This material may constitute less than 100% of growthmedia. In some embodiments the wastewater is supplemented with biotin.In some embodiments this material is supplemented with ammonium gas. Insome embodiments this material is supplemented with di-ammoniumphosphate. In some embodiments this material is supplemented withammonium nitrate. In some embodiments this material is supplemented withyeast extract. In some embodiments this material is supplemented withpotassium nitrate. In some embodiments this material is supplementedwith potassium phosphate. In some embodiments this material issupplemented with calcium chloride. In some embodiments this material issupplemented with magnesium sulfate. In some embodiments this materialis supplemented with nitrates. In some embodiments this material issupplemented with ammonium salts. In some embodiments this material issupplemented with the aforementioned plant ingredients described herein.In some embodiments the material is supplemented with aforementionedwaste streams or co-products described herein.

In some embodiments the potato processing wastewater chemical oxygendemand (COD) concentration measures between about 500 mg/L and about300,000 mg/L, between about 2,000 mg/L and about 200,000 mg/L, betweenabout 2,000 mg/L and about 100,000 mg/L, between about 2,000 mg/L andabout 50,000 mg/L, between about 2,500 mg/L and about 25,000 mg/L, orbetween about 2,000 mg/L and about 10,000 mg/L.

In certain embodiments, corn stillage is captured, optionallysterilized, optionally supplemented, and injected into the bioreactorsrunning the fermentations of the present invention. Thin stillage thatcan be used has an average of 85,000 mg/L COD and about 5,000 mg/L oftotal nitrogen. This material may constitute 100% of the growth mediadescribed herein. This material may constitute less than 100% of growthmedia. In some embodiments the wastewater is supplemented with biotin.In some embodiments this material is supplemented with ammonium gas. Insome embodiments this material is supplemented with di-ammoniumphosphate. In some embodiments this material is supplemented withammonium nitrate. In some embodiments this material is supplemented withyeast extract. In some embodiments this material is supplemented withpotassium nitrate. In some embodiments this material is supplementedwith potassium phosphate. In some embodiments this material issupplemented with calcium chloride. In some embodiments this material issupplemented with magnesium sulfate. In some embodiments this materialis supplemented with nitrates. In some embodiments this material issupplemented with ammonium salts. In some embodiments this material issupplemented with the aforementioned plant ingredients described herein.In some embodiments the material is supplemented with aforementionedwaste streams or co-products described herein.

Shelf-Stable Food Ingredient

In another aspect, provided herein are shelf-stable protein foodingredients. The shelf-stable protein food ingredients comprise culturedfungal biomass and a limited amount of water. The shelf-stable proteinfood ingredients can be prepared according to the methods above. Theshelf-stable food ingredients are designed to be a versatile, primarilytextured, consistent sized material that is dry, storable, and optimizedfor ease of use in an end product. Provided herein are exemplarydetailed characterizations of the shelf stable protein food ingredientsprovided herein.

Texture: Texture of the ingredient is important when being used as ameat analogue or a meat extension agent. The filamentous nature of thefungi described herein provides compacted and aligned fibers that mimicmuscle in some ways. Texture was analyzed using a rheometer with a 25 mmdiameter cylinder probe. The TCP was hydrated for 30 mins at a 1/1.75w/w ratio of TCP/water. The cross head speed was set to 100 mm/min⁻¹with a max peak stress of 10 kg and a distance between the two supportsof 13 mm. The results were averages of 20 treatments.

Chewiness: Chewiness was analyzed using the described methods in “BreeneW M, Application of texture profile analysis to instrumental foodtexture evaluation. J Texture Stud 6:53-82 (1975)” using hydrated TCP ofthe present invention. Chewiness was typically between about 0.5 kg andabout 15 kg, between about 1 kg and about 12 kg, between about 2 kg andabout 10 kg, or between about 4 kg and about 8 kg.

Cohesiveness: Cohesiveness was determined by taking the dry shelf-stableingredient of the present invention, hydrating it at a 1/1.75 ratio w/wof dry product to water, treating the material with the followingprocess, and analyzing texture residues. After hydration, the materialwas subsequently pressurized, dispersed, and dried. Cohesiveness wastypically between about 20% and about 90%, between about 30% and about80%, or between about 40% and about 60%.

Springiness: Springiness was analyzed using the described methods in“Breene W M, Application of texture profile analysis to instrumentalfood texture evaluation. J Texture Stud 6:53-82 (1975)” using hydratedTCP of the present invention. Springiness was typically between about15% and about 99%, between about 20% and about 85%, between about 40%and about 70%, or between about 40% and about 60%.

Transversal cutting strength: Cutting strength was determined by using acutting probe (7.5 mm×38.3 mm) with a 2 kg maximum peak stress. Cuttingstrength helps with determining bite resistance, shear, and texture asit relates to maceration in the human mouth.

Longitudinal cutting strength: Cutting strength was determined by thesame methods used for the transversal cutting strength tests.

Protein: The protein content of the ingredients described hereincomprise at least 5% total protein. In certain embodiments, theshelf-stable protein food ingredients comprise protein in an w/w amountof 5-100%, 5-75%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, or5-15%. The amino acid profile may comprise of a number of amino acids.Total protein content of an ingredient can be determined by manydifferent methods including but not limited to AOAC Internationalmethods 990.03 and 992.15. In some embodiments the ingredient containsthe amino acids Methionine, Cystine, Lysine, Phenylalanine, Leucine,Isoleucine, Threonine, Valine, Histidine, Arginine, Glycine, Asparticacid, Serine, Glutamic acid, Proline, Hydroxyproline, Alanine, Tyrosine,and Tryptophan, Taurine, and others. The ingredient contains all of theessential amino acids for the human diet.

Fat: The fat composition of the ingredient described herein comprisesmostly mono- and polyunsaturated fats and can be very low in saturatedfats. In some embodiments the total fat content comprises a w/w amountbetween about 1% and about 80%, between about 1% and about 70%, betweenabout 1% and about 60%, between about 1% and about 50%, between about 2%and about 40%, between about 3% and about 30%, between about 5% andabout 30%, between about 6% and about 20%, between about 7% and about15%, between about 8% and 13%, between about 10% and about 14% by weightof total fat. Fats are primarily monounsaturated and polyunsaturatedfats. In some embodiments saturated fats are between 0% and about 40%,between 0% and about 30%, between about 5% and about 20%, and betweenabout 10% and about 20% of the total fat content. In some embodimentsunsaturated fats are between about 10% and about 100%, between about 20%and about 100%, between about 30% and about 100%, between about 40% andabout 100%, between about 60% and about 90%, or between about 70% andabout 80% of the total fat content.

Fiber: In certain embodiments, the shelf-stable protein food ingredientsdescribed herein are naturally high in fiber. This can be positiveaspect of this type of meat like product. AOAC method 991.43 can be usedto determine the fiber content of the ingredients described herein. Insome embodiments, fiber content is between about 5% and about 60%,between about 10% and about 50%, between about 15% and about 40%,between about 20% and about 40%, or between about 30% and about 40%.

Vitamins: In certain embodiments, the shelf-stable protein foodingredients comprise a range of water-soluble B vitamins sometimesconsisting of thiamin, riboflavin, niacin, pyridoxine, pantothenic acid,folic acid, biotin, and others.

Minerals: In certain embodiments, the shelf-stable protein foodingredients comprise calcium, phosphorous, magnesium, iron, zinc,sodium, manganese and potassium. Calcium is typically in an amount of200 mg/kg or more. In some embodiments, calcium is between about 500mg/kg and about 3000 mg/kg, between about 1000 mg/kg and about 2500mg/kg, between about 1250 mg/kg and about 2000 mg/kg, and between about1500 mg/kg and 2000 mg/kg. Phosphorous is typically in an amount of 200mg/kg or more In some embodiments phosphorous is between about 500 mg/kgand about 2500 mg/kg, between about 500 mg/kg and about 2000 mg/kg,between about 750 mg/kg and about 1500 mg/kg, and between about 800mg/kg and 1200 mg/kg. Potassium is typically in an amount of 100 mg/kgor more. In some embodiments, potassium is between about 1000 mg/kg andabout 8000 mg/kg, between about 2000 mg/kg and about 6000 mg/kg, betweenabout 2500 mg/kg and about 5000 mg/kg, and between about 3000 mg/kg and4500 mg/kg. Sodium is in an amount of 20 mg/kg or more. In someembodiments sodium is between about 20 mg/kg and about 1500 mg/kg,between about 50 mg/kg and about 400 mg/kg, between about 100 mg/kg andabout 300 mg/kg, between about 150 mg/kg and about 250 mg/kg, betweenabout 175 mg/kg and about 225 mg/kg. Magnesium is in an amount of 200mg/kg or more. In some embodiments, magnesium is between about 500 mg/kgand about 3000 mg/kg, between about 1000 mg/kg and about 2500 mg/kg,between about 1250 mg/kg and about 2000 mg/kg, and between about 1500mg/kg and about 2000 mg/kg. Iron is typically in an amount of 1 mg/kg ormore. In some embodiments iron is between about 2 mg/kg and about 100mg/kg, between about 5 mg/kg and about 80 mg/kg, between about 10 mg/kgand about 50 mg/kg, or between about 20 mg/kg and 40 mg/kg. Zinc is inan amount of 20 mg/kg or more. In some embodiments zinc is between about20 mg/kg and about 1500 mg/kg, between about 100 mg/kg and about 600mg/kg, between about 200 mg/kg and about 500 mg/kg, between about 300mg/kg and about 500 mg/kg, between about 350 mg/kg and about 450 mg/kg.

Water holding capacity (WHC): In certain embodiments, the shelf-stableprotein food ingredients described herein has a WHC significantly higherthan that of traditional TVP, that of meat, and that of plantingredients. WHC is analyzed by fully removing all moisture from theingredient, weighing the ingredient, then fully hydrating theingredient, then removing surface moisture, then weighing again. Allsamples are analyzed in quadruplicate and the average is taken. We haverecorded the WHC of the TCP as being as high as 6,743 g/kg. In preferredembodiments the WHC of the TCP ingredient is between about 2,000 g/kgand about 7,000 g/kg. In some embodiments the WHC is between about 2,000g/kg and about 6,000 g/kg, between about 2,000 g/kg and about 5,000g/kg, between about 3,000 g/kg and about 5,000 g/kg, between about 3,500g/kg and about 4,500 g/kg, or between about 4,000 g/kg and about 5,000g/kg.

Particle size: In certain embodiments, the shelf-stable protein foodingredients are manipulated during the processing of the ingredient tohave specific particle sizes. Certain particle sizes work best forcertain applications. Particle sizes usually range from a fine powder to1000 cm sheets. In certain embodiments the particle size is betweenabout 2 mm and about 40 mm, between about 2 mm and about 30 mm, betweenabout 2 mm and about 20 mm, between about 2 mm and about 15 mm, betweenabout 2 mm and about 10 mm, between about 2 mm and about 8 mm, betweenabout 2 mm and about 6 mm, between about 3 mm and about 10 mm, betweenabout 3 mm and about 7 mm, between about 4 mm and about 6 mm, betweenabout 4 mm and about 10 mm, between about 5 mm and about 50 mm, betweenabout 5 mm and about 40 mm, between about 5 mm and about 20 mm, betweenabout 5 mm and about 10 mm, between about 6 mm and about 50 mm, betweenabout 6 mm and about 40 mm, between about 6 mm and about 30 mm, betweenabout 6 mm and about 20 mm, between about 6 mm and about 10 mm, between6 mm and about 10 mm, between about 7 mm and about 20 mm, between about7 mm and about 15 mm, between about 7 mm and about 12 mm, between about7 mm and about 10 mm. In preferred embodiments the particles are 3 mm, 4mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, and 12 mm.

In some embodiments the particle size is measured with “D-values”. D10,D50, and D90 represent the percentage of particles less than a certainsieve size (10%, 50%, and 90% respectively). In particular, D10indicates a diameter at which 10% of the mass of a sample is inparticles less than the indicated diameter. D50 indicates a diameter atwhich 50% of the mass of a sample is in particles less than theindicated diameter. D90 indicates a diameter at which 90% of the mass ofa sample is in particles less than the indicated diameter. In certainembodiments the TCP described herein has a D50 of about 5 mm, a D50 ofabout 6 mm, a D50 of about 7 mm, a D50 of about 4 mm, a D50 of about 8mm, a D50 of about 9 mm, a D50 of about 10 mm, a D50 of about 11 mm, aD50 of about 15 mm, a D10 of about 2 mm, a D10 of about 3 mm, a D10 ofabout 4 mm, a D10 of about 5 mm, a D10 of about 6 mm, a D10 of about 7mm, a D10 of about 8 mm, a D10 of about 9 m, a D10 of about 10 mm, a D10of about 11 mm, a D10 of about 12 mm, a D90 of about 2 mm, a D90 ofabout 50 mesh, a D90 of about 70 mesh, a D90 of about 100 mesh, a D90 ofabout 1 mm, a D90 of about 2 mm, a D90 of about 3 mm, a D90 of about 4mm, a D90 of about 5 mm, a D90 of about 6 mm, a D90 of about 7 mm, a D90of about 8 mm, or a D90 of about 9 mm.

In certain embodiments, the term “about” indicates +/−1 mm. In certainembodiments, the D10, D50, and D90 values are exact, or exact within thetolerance range of the measurement technique. Particle size can bemeasured according to standard techniques, for instance sieve analysis.

Color: In certain embodiments, the shelf-stable protein food ingredientshave a naturally white/off white color. In certain embodiments, theshelf-stable protein food ingredients have an off-white consistentcolor. In certain embodiments, the shelf-stable protein food ingredientshave a tan color. In some embodiments, the material is affected byaforementioned substrate components. Sometimes this color is caramel,red, pink, green, brown, yellow, and other hues. In some embodiments,the color of the shelf-stable protein food ingredients described hereinis affected by the plant ingredients that are sometimes combined withthe mycelium. Sometimes this color is caramel, red, pink, green, brown,yellow, orange, and other hues.

In some embodiments described herein additional ingredients areintegrated into the fermentation process and some concentrations ofthese additional ingredients remain in the dehydrated product describedherein. These supplementary ingredients may alter the nutritionalprofile, texture, or other properties of the dry ingredients.

In some embodiments, additional ingredients are combined with theshelf-stable protein food ingredients described herein to form a complexingredient with enhanced functional properties. The ingredients may beselected from the following: pea fiber, other plant fibers, gum arabic,natural flavors, texturized pea protein, texturized wheat protein,texturized soy protein, soy protein, wheat starch, wheat protein, peaprotein, spices, safflower oil, sunflower oil, olive oil, other oils,oat bran, oat flour, legumes, beans, lentils, lentil powder, beanpowder, pea powder, yeast extract, nutritional yeast (immobilized driedyeast), molasses, honey, cane sugar, mushroom powder, white buttonmushroom powder, shiitake mushroom powder, chickpeas, bamboo fiber,cellulose, isolated oat product, isolated pea product, pea protein, riceprotein, fermented rice extract, corn starch, potato starch, kombuextract, algae, potato protein, albumin, pectin, silicone dioxide, foodstarch, mixed tocopherols (vitamin E), coconut oil, sunflower oil,safflower oil, rapeseed oil, canola oil, dextrose, vegetable glycerin,dried yeast, citrus extract, citrus fiber, beet pulp, beet juice, beetjuice extract, turmeric, mushroom extract, shiitake mushroom stems,shiitake mushrooms, white button mushrooms, tofu, soy fiber, soyhydrolysate, yeast extract and seaweed, natural flavorings, or anycombination thereof.

In some embodiments the ingredients described herein is combined withmaterials known to enhance texture, flavor, palatability, shelf life,stability, and other properties known to people with skill in the art ofprotein ingredients. These materials can be but are not limited toalbumin, pectin, silicone dioxide, zinc gluconate, vitamin B12,maltodextrin, niacin, sodium ascorbate, pyridoxine hydrochloride,tetrasodium pyrophosphate, calcium carbonate, sodium alginate, alginate,trisodium phosphate, calcium acetate, methylcellulose, cellulose, bamboocellulose, annatto, acetic acid, sodium nitrite, sodium benzoate, soylecithin, or any combination thereof.

In certain embodiments the shelf-stable protein food ingredientdescribed herein is milled into flour using conventional millingequipment. This flour provides the same nutritional profileaforementioned here while also having properties including but notlimited to, gumming properties, tacking properties, enhanced nutrition,high fiber, dough like properties, and other flour like properties thatlend themselves to being an effective flour replacement or enhancer. Insome embodiments the aforementioned flour is combined with plant basedflours such as corn flour, wheat flour, sorghum flour, rye flour, milletflour, quinoa flour, and other flours. In some embodiments theaforementioned flour is used in products like a protein bar, bread,pasta, and other flour

In some embodiments plant or mushroom biomass is combined with theshelf-stable protein food ingredients described herein. The addedproperties of the plant/mushroom biomass enhances the product. Suchenhancements are but are not limited to color, texture, density, flavor,cooking properties, aesthetics, nutrition, etc. Such plants andmushrooms can be but are not limited to; beet root (Beta vulgaris), kingoyster mushroom (Pleurotus eryngii), oyster mushroom (Pleurotusostreatus), shiitake mushroom (Lentinula edodes), or portabello mushroom(Agaricus bisporus).

Food Products

In another aspect, provided herein are food products comprising theshelf-stable protein food ingredients. The food products comprise theshelf-stable protein food ingredients and one or more additional foodingredients. In certain embodiments, the shelf-stable protein foodingredients are mixed with fat, carbohydrate, meat, plant, or acombination thereof. In certain embodiments, the shelf-stable proteinfood ingredients are mixed with plant protein to form food products. Incertain embodiments, the shelf-stable protein food ingredients are mixedwith meat protein to form food products.

The meat can be any meat deemed suitable by the practitioner of skill.In certain embodiments, the meat is selected from the group consistingof beef, pork, chicken, turkey, lamb, fish, venison, bison, andcombinations thereof. In certain embodiments, a shelf-stable proteinfood ingredient is mixed or layered with beef. In certain embodiments, ashelf-stable protein food ingredient is mixed or layered with pork. Incertain embodiments, a shelf-stable protein food ingredient is mixed orlayered with chicken. In certain embodiments, a shelf-stable proteinfood ingredient is mixed or layered with turkey. In certain embodiments,a shelf-stable protein food ingredient is mixed or layered with lamb. Incertain embodiments, a shelf-stable protein food ingredient is mixed orlayered with fish. In certain embodiments, a shelf-stable protein foodingredient is mixed or layered with crab. In certain embodiments, ashelf-stable protein food ingredient is mixed or layered with lobster.In certain embodiments, a shelf-stable protein food ingredient is mixedor layered with venison. In certain embodiments, a shelf-stable proteinfood ingredient is mixed or layered with bison.

In certain embodiments, the resulting composition is further mixed withany other food ingredient deemed suitable to the person of skill. Incertain embodiments, the additional ingredient is selected from thegroup consisting of starches, oils, fats, grains, isolates, fibers,plants, algae, mushrooms, and combinations thereof. In certainembodiments, the additional ingredient is selected from the groupconsisting of pea fiber, other plant fibers, gum arabic, naturalflavors, texturized pea protein, texturized wheat protein, texturizedsoy protein, soy protein, wheat starch, wheat protein, pea protein,spices, safflower oil, sunflower oil, olive oil, other oils, oat bran,oat flour, legumes, beans, lentils, lentil powder, bean powder, peapowder, yeast extract, nutritional yeast (immobilized dried yeast),molasses, honey, cane sugar, mushroom powder, white button mushroompowder, shiitake mushroom powder, chickpeas, bamboo fiber, cellulose,isolated oat product, isolated pea product, pea protein, rice protein,fermented rice extract, corn starch, potato starch, kombu extract,algae, potato protein, albumin, pectin, silicone dioxide, food starch,mixed tocopherols (vitamin E), coconut oil, sunflower oil, saffloweroil, rapeseed oil, canola oil, dextrose, vegetable glycerin, driedyeast, citrus extract, citrus fiber, beet pulp, beet juice, beet juiceextract, turmeric, mushroom extract, shiitake mushroom stems, shiitakemushrooms, white button mushrooms, tofu, soy fiber, soy hydrolysate,yeast extract and seaweed, natural flavorings, or any combinationthereof.

Generally, the food products comprise fungal biomass in an amount of atleast 5% w/w. The food products further comprise at least one meat in anamount of at least 10% w/w. In certain embodiments, when more than onemeat is present the total amount of meat is at least 10% w/w.Advantageously, the food products can be prepared according to themethods above.

In certain embodiments, the food products comprise at least 5% w/wfungal biomass and at least 10% w/w meat. In certain embodiments, thefood products comprise at least 5% w/w fungal biomass and at least 10%w/w meat. In certain embodiments, the food products comprise at least10% w/w fungal biomass and at least 10% w/w meat. In certainembodiments, the food products comprise at least 15% w/w fungal biomassand at least 10% w/w meat. In certain embodiments, the food productscomprise at least 20% w/w fungal biomass and at least 10% w/w meat. Incertain embodiments, the food products comprise at least 10% w/w fungalbiomass and at least 10% w/w meat. In certain embodiments, the foodproducts comprise at least 25% w/w fungal biomass and at least 10% w/wmeat. In certain embodiments, the food products comprise at least 30%w/w fungal biomass and at least 10% w/w meat. In certain embodiments,the food products comprise at least 35% w/w fungal biomass and at least10% w/w meat. In certain embodiments, the food products comprise atleast 40% w/w fungal biomass and at least 10% w/w meat. In certainembodiments, the food products comprise at least 45% w/w fungal biomassand at least 10% w/w meat. In certain embodiments, the food productscomprise at least 50% w/w fungal biomass and at least 10% w/w meat.

In certain embodiments, the food products comprise at least 5% w/wfungal biomass and at least 20% w/w meat. In certain embodiments, thefood products comprise at least 10% w/w fungal biomass and at least 20%w/w meat. In certain embodiments, the food products comprise at least20% w/w fungal biomass and at least 20% w/w meat. In certainembodiments, the food products comprise at least 15% w/w fungal biomassand at least 20% w/w meat. In certain embodiments, the food productscomprise at least 20% w/w fungal biomass and at least 20% w/w meat. Incertain embodiments, the food products comprise at least 20% w/w fungalbiomass and at least 20% w/w meat. In certain embodiments, the foodproducts comprise at least 25% w/w fungal biomass and at least 20% w/wmeat. In certain embodiments, the food products comprise at least 30%w/w fungal biomass and at least 20% w/w meat. In certain embodiments,the food products comprise at least 35% w/w fungal biomass and at least20% w/w meat. In certain embodiments, the food products comprise atleast 40% w/w fungal biomass and at least 20% w/w meat. In certainembodiments, the food products comprise at least 45% w/w fungal biomassand at least 20% w/w meat. In certain embodiments, the food productscomprise at least 50% w/w fungal biomass and at least 20% w/w meat.

In certain embodiments, the food products comprise at least 5% w/wfungal biomass and at least 30% w/w meat. In certain embodiments, thefood products comprise at least 10% w/w fungal biomass and at least 30%w/w meat. In certain embodiments, the food products comprise at least20% w/w fungal biomass and at least 30% w/w meat. In certainembodiments, the food products comprise at least 15% w/w fungal biomassand at least 30% w/w meat. In certain embodiments, the food productscomprise at least 20% w/w fungal biomass and at least 30% w/w meat. Incertain embodiments, the food products comprise at least 20% w/w fungalbiomass and at least 30% w/w meat. In certain embodiments, the foodproducts comprise at least 25% w/w fungal biomass and at least 30% w/wmeat. In certain embodiments, the food products comprise at least 30%w/w fungal biomass and at least 30% w/w meat. In certain embodiments,the food products comprise at least 35% w/w fungal biomass and at least30% w/w meat. In certain embodiments, the food products comprise atleast 40% w/w fungal biomass and at least 30% w/w meat. In certainembodiments, the food products comprise at least 45% w/w fungal biomassand at least 30% w/w meat. In certain embodiments, the food productscomprise at least 50% w/w fungal biomass and at least 30% w/w meat.

In certain embodiments, the food products comprise at least 5% w/wfungal biomass and at least 40% w/w meat. In certain embodiments, thefood products comprise at least 10% w/w fungal biomass and at least 40%w/w meat. In certain embodiments, the food products comprise at least20% w/w fungal biomass and at least 40% w/w meat. In certainembodiments, the food products comprise at least 15% w/w fungal biomassand at least 40% w/w meat. In certain embodiments, the food productscomprise at least 20% w/w fungal biomass and at least 40% w/w meat. Incertain embodiments, the food products comprise at least 20% w/w fungalbiomass and at least 40% w/w meat. In certain embodiments, the foodproducts comprise at least 25% w/w fungal biomass and at least 40% w/wmeat. In certain embodiments, the food products comprise at least 30%w/w fungal biomass and at least 40% w/w meat. In certain embodiments,the food products comprise at least 35% w/w fungal biomass and at least40% w/w meat. In certain embodiments, the food products comprise atleast 40% w/w fungal biomass and at least 40% w/w meat. In certainembodiments, the food products comprise at least 45% w/w fungal biomassand at least 40% w/w meat. In certain embodiments, the food productscomprise at least 50% w/w fungal biomass and at least 40% w/w meat.

In certain embodiments, provided herein is a meat analogue from thebiomass of Tuber melanosporum as a “truffle burger”.

In some embodiments the ingredient described herein is combined withseaweed biomass. In certain embodiments the ingredient described hereinis combined with algae.

Detailed Description of the Figures

FIG. 1 provides an overview of an embodiment of the fermentation processused to produce the biomass described herein

Block 1 describes the substrate feedstock used for the fermentation andthe injection of said substrate into the reactor post sterilization. Thesubstrate can be a waste stream, plant hydrolysate, plant material,nutrient salts, sugars, starches, fatty acids, proteins, and othernutrients.

Block 2 describes the introduction of the filamentous fungi describedherein into the substrate.

Block 3 describes the fermentation parameters described herein.

Blocks 4 a. and 5 a. describe an optional intermittent substrateintroduction and harvesting fermentation operation strategy.

Blocks 4 b. and 5 b. describe an optional continuous substrateintroduction and harvesting fermentation operation strategy.

Block 4 c. describes an optional batch fermentation operation strategy.

FIG. 2 provides an overview of some methods for the processing of thefungal biomass slurry into the textured cultured protein (TCP) describedherein

Blocks 1 a.-1 d. disclose the raw biomass slurry of the fermentationsdescribed herein. The slurry includes but is not limited to biomassfermented with wastewater, co-products, and/or side streams from foodand beverage processing, biomass fermented with conventionalfermentation substrates, and biomass fermented with plant basedingredients. The biomass may be rinsed to remove residual substrate. Thebiomass may have the aformentioned plant ingredients integrated at thispoint. The material may be pasteurized post-harvest from thefermentation.

Blocks 2 a.-2 e. disclose the steps of processing the biomass to removemoisture via de-watering methods and the option to integrate theplant-based ingredients described herein. The supernatant may optionallybe recovered for further purification.

Blocks 3 a.-3 d. disclose the steps of sizing the particles describedherein. Different isolated particle sizes as well as combinations ofparticle sizes lend themselves to different applications in foodproducts.

Blocks 4 a.-5 c. disclose optional pasteurization steps where particlesand pieces are pasteurized prior to, during, or after dehydration.

Block 4 a. describes the optional steps of freezing the pieces of TCP.

Blocks 5 a.-5 c. disclose the dehydration and desiccation steps for theremoval of moisture from the biomass to make the dry and shelf stableingredient described herein.

Block 6. Discloses the optional combination of other ingredients withthe dried pieces to form a composition.

Block 7. discloses the dry ingredient described herein.

FIG. 3 provides an overview of one embodiment of the fungal biomassfermentation described herein

Block 1-4 discloses some possible plant materials to be processed intosubstrate. The plant material can be sourced as waste streams from thefood and beverage industries.

Block 5 discloses the step of processing the plant materials intosubstrate of the fermentation. The plant material is soaked in 100 Cwater and agitated for 10-120 minutes. The hydrolyzed plant material isseparated from the liquid hydrolysate via filtration.

Block 6 discloses the addition of supplements to balance thecarbon/nitrogen rations and overall nutritional profile of thesubstrate. Supplements can be but are not limited to, ammoniumphosphate, potassium nitrate, calcium sulfate, glucose, sucrose, or anycombination thereof.

Block 7 closes the requirement of the substrate sterility. The majorityof the substrate is heat sterilized, however select ingredients such asthe vitamins (for example, vitamin B12 and vitamin B7; (biotin)) arefiltered and injected separately.

Block 8 discloses the transfer of the media into the reactor.

Block 9 discloses the methods for inoculation. Liquid seed cultures ofmycelium may be transferred into the reactor, high loads of spores canbe injected into the substrate, or a volume of biomass/substrate can bein the reactor already, propagated as an inoculum. It is a facetdescribed herein to use fungi of the Phyla Ascomycota and Zygomycotaincluding but not limited to; Aspergillus oryzae, Rhizopus oryzae,Fusarium graminareum, Cordyceps militaris, Cordyceps sinensis, Tubermelanosporum, Tuber magnatum, Pennicillium camemberti, Neurosporaintermedia or Xylaria hypoxion.

Block 10 discloses the addition of a species of fungi as the organism ofthe fermentation.

Block 11 discloses the fermentation method. Temperature is maintained at20-34° C., aeration is increased over time using both ambient gasconcentrations as well as pure oxygen ranging from 0.03-2 vvm (volume ofair per volume of medium per minute). PH is maintained in a state thatis ideal for filamentous fungi. 3-8 is the range described herein.

Block 12 discloses the pasteurization of the mycelium. This destroys theviability of the fungus to continue growing and extracts much of the RNAthat in undesirable in cellular protein products.

Block 13 discloses the extraction of the biomass and substrate mixturefrom the bioreactor.

Block 14 discloses the step of de-watering the fungal mycelium. The highmoisture retention properties of the mycelium make it necessary toremove some liquid for ideal moisture contents that mimic meat.

Block 15 discloses the remainder of de-watering. This supernatantcontains enzymes, acids, lipids, and other valuable extracellularcomponents.

Block 16 discloses the end multi-use fungal biomass material describedherein. This is the material that gets used in the blended meats, theblended plant, and the TCP food products.

FIG. 4 provides an overview of optional applications of the ingredientsdescribed herein

Block 17 Discloses the raw food material described herein.

Block 18 disclosed the blending of the fungal biomass into meats toproduce hybrid meat/fungi food products.

Block 19 discloses some but not all of the meats and meat products thematerial is integrated into.

Block 20 discloses the addition of plant and mushroom biomass into theblended fungi/meat products as well as into the fungi based meatalternatives.

Block 21 discloses the use of the material described herein in meatalternative products.

Block 22 discloses some but not all methods for preparation andtexturization of the material described herein.

Block 23 discloses some ways in which the meat analogue products arefurther prepared and cooked.

FIG. 5A provides a close-up photograph on an exemplary TCP providedherein. FIG. 5B provides a photograph of an exemplary TCP providedherein on a serving plate.

EXAMPLES

Below the methods and compositions are further described in examples.These examples are in no way described to limit the present embodimentsor their contents.

Example 1

Spores from Rhizopus oryzae were plated on agar containing vesselsconsisting of 15 g/l sucrose, 3 g/l Na3 citrate, 5 g/l KH2PO4, 2 g/lNH4NO3, 0.2 g/l MgSO4, 1 g/l CaSO4, 0.005 g/l Zn SO4, 0.001 g/lFe(NH4)2(SO4)2, 0.00025 g/l CuSO4, 0.0001 g/l MnSO4, 0.0025 g/l biotin,and 15 g/l agar.

The cultures were incubated at 28° C. for 3 days to encourage maximumsporulation. The air/agar interface supplies aerial hyphae withnutrients embedded in the substrate while exposing hyphae to oxygen.Generous airflow was provided to the culture vessels. The spores werecollected in a sterile polypropylene vessel.

The 17 L reactor was prepared by sterilization via 130° C. steam andintroducing autoclaved substrate. The reactor substrate consisted of 17liters DI water, 30 g/l light malt extract prepared from thehydrolyzation and filtration of malted barley, 10 g/l glucose, 5 g/1yeast extract, 0.5 g/l NH₄H₂NO₃, 0.2 g/l MgSO₄, 3 g/l safflower oil, and0.0025 g/l biotin.

These spores were then introduced to the 17 l reactor at theconcentration of 50,000/ml. The reactor was incubated at 29° C. with anincreasing aeration starting at 0.1 vvm and reaching 0.8 vvm atmosphericair after 24 hours. The fermentation was complete after 48 hours.

The end substrate/biomass mixture was pressed in a porous cubic frame tode-water the biomass down to 68% for use in aforementioned foodproducts. Samples were taken of the biomass and dried out at 101° C. Itwas determined via triplicate sampling that the reactor yielded 17 g/lof dry biomass.

Example 2

Aerobic fermentation was carried out in pulse feeding mode. 17 l DIwater, 30 g/l light malt extract prepared from the hydrolyzation ofspent malted barley, 10 g/l glucose, 5 g/l yeast extract, 0.5 g/lNH₄H₂NO₃, 0.2 g/l MgSO₄, 3 g/l safflower oil, and 0.0025 g/l biotin wasautoclaved and added to the sterile reactor. 150 ml of a 2 day oldliquid culture of Cordyceps militaris mycelium grown on 25 g/l sucrose,3 g/l Na₃ citrate, 5 g/l KH₂PO₄, 2 g/l NH₄NO₃, 0.2 g/l MgSO₄, 1 g/lCaSO₄, 0.005 g/l Zn SO₄, 0.001 g/l Fe(NH₄)₂(SO₄)₂, 0.00025 g/l CuSO₄,0.0001 g/l MnSO₄, and 0.0025 g/l biotin. The reactor was maintained at apH above 4.5 by dripping NaOH and held at 25 C with an increasingambient air aeration starting at 0.1 vvm and reaching 0.8 vvm after 24hours. 20 g/l light malt, 10 g/l glucose, and 20 g/l yeast extract wasautoclaved and injected into the reactor on hour 48 of the incubation.This substrate supplementation was repeated on hour 96. The growth phaseended on hour 143. The substrate/biomass mixture was pressed in a porouscubic frame and de-watered down to 65% water content. The supernatantwas disposed. The pulse feeding strategy yielded 41 g/l of dry biomass.

Example 3

Aerobic fermentation was carried out in pulse feeding/pulse harvestmode. 17 l DI water, 30 g/l potato extract prepared from thehydrolyzation of potato skin, 10 g/l glucose, 2 g/l yeast extract, 0.5g/l NH₄H₂NO₃, 0.2 g/l MgSO₄, 3 g/l safflower oil, and 0.0025 g/l biotinwas autoclaved and added to the sterile reactor. 50,000 Aspergillusoryzae spores/l were added to the substrate. The reactor was maintainedat a pH of 4.5 by dripping NaOH into the substrate as needed. Thereactor was maintained at 29 C with an increasing ambient air aerationstarting at 0.1 vvm and reaching 0.8 vvm after 24 hours. After 48 hours85% of the substrate biomass mixture was removed. A sterile solution of14 l DI water, 30 g/l potato extract, 10 g/l glucose, 2 g/l yeastextract, 0.5 g/l NH₄H₂NO₃, 0.2 g/l MgSO₄, 3 g/l safflower oil, and0.0025 g/l biotin was added filling the reactor back up to almost 17 l.The fermentation continued for another 24 hours before the extractionand re-supplementation process was repeated at hour 72. This was thenrepeated a third time at hour 96. The final harvest of the entire volumeof the reactor occurred at hour 120. Biomass was removed and de-watereddown to 65% using pressure in a cubic porous frame for furtherprocessing into product samples. Biomass yield samplings were carriedout in triplicates at each biomass extraction point (hr 48, hr 72, hr96, and hr 120). Hour 48 had an average of 16.3 g/l dry biomass, hour 72had an average of 15.7 g/l, hour 96 had an average of 16.1 g/l, and hour120 had an average of 14.2 g/l.

Example 4

Biomass of Neurospora intermedia (500 g) at 80% water content culturedfor 72 hours at 29 C with 0.5 v/v/m of aeration in a 300 1 internal loopbioreactor at a pH >4.5 on 30 g/l light malt extract, 10 g/l glucose, 20g/l yeast extract, 0.5 g/l NH₄H₂NO₃, 0.2 g/l MgSO₄, 3 g/l safflower oil,and 0.0025 g/l biotin added to a KitchenAid mixer with 50% (w/w) grassfed ground chuck beef (500 g) for a total of 1000 g of meat/myceliummixture. The mixture was blended on low for 8 minutes until homogenousin texture and color. The mixture was removed and hand pressed into 5circular patties 1.5″ in height x 6″ in diameter. Two patties weregrilled over a propane burner on a “medium” propane flow rate untilconsidered cooked to a point of classification as “medium”. Two pattieswere pan seared on medium heat until slightly blackened and long enoughto be considered “medium”. Two patties were baked at 425 F for 15minutes. Two patties were flame cooked over charcoal until they could beconsidered “medium”. Two patties were pan seared until they could beconsidered “medium rare”. One control patty for each cooking method ofpure chuck ground beef was cooked in the same fashion as the blendedpatties as a control.

Example 5

Biomass of Aspergillus oryzae (300 g) cultured in supplemented potatohydrolysate was dried in a dehydrator and pulverized into a powder usinga mortar and pestle. The powder was mixed with mixed with whole wheatflower (50 g). The mixture was hydrated with 2 cups of whole milk andcombined with 3 eggs. The mixture was blended together with ½ stick ofbutter until malleable dough was created. The dough was rolled into 2″balls and fried. Powdered sugar was added on top of the balls.

Example 6

Asexually sporulating cultures of Neurospora intermedia were used toinoculate 8 baffled shaker flasks with a working volume of 200 ml and a0.2 micron filter patch embedded in the cap. The media contained in theflasks contained 30 g/l sucrose, 3 g/l Na3 citrate, 2 g/l KH2PO4, 2 g/lNH4NO3, 0.2 g/l MgSO4, 1 g/l CaSO4, 0.005 g/l Zn SO4, 0.001 g/lFe(NH4)2(SO4)2, 0.00025 g/l CuSO4, 0.0001 g/l MnSO4, 0.0025 g/l biotin,and 15 g/l agar agar. These flasks were incubated and agitated for 24hours at 100 rpm and 29° C. on a incubated shaker table.

A 100 liter internal loop airlift bioreactor (ILAB) was filled to an 80L working volume with DI water. Using a magnetically coupled agitatorfor agitation, 45 g/L potato flour, 2 g/L yeast extract, 0.5 g/LNH₄H₂NO₃, 0.0025 g/l biotin was successively added to the vessel untilhomogenous. The media was sterilized with steam in place methods (125°C. for 20 minutes). The eight 24-hour old baffled flask starter cultureswere aseptically injected into the 100 L reactor. The reactor wasmaintained at 31° C. The reactor was maintained at a pH of 4.5 bydripping NaOH into the substrate as needed. Compressed air was injectedthrough a sintered steel sparger with a porosity of 0.2 microns in theriser section of the ILAB at a flow rate of 0.1 vvm. The flow rate wasincreased to 1.0 vvm over the first 48 hours.

These conditions were maintained for 72 hours.

The biomass slurry was filtered using nylon mesh filter sacks to removethe supernatant leaving a high moisture biomass. The high moisturebiomass was centrifuged to further reduce the moisture content down to75%. The reduced moisture biomass was shredded into particles less than0.25″ by using a shaft mounted 5 blade shredder at 500 rpm. Particleswere sieved to separate the particle sizes. Particles were dehydrated at50° C. for 120 minutes until bone dry with a moisture content of 4%. Thematerial was packaged and stored.

Example 7

A 48 hour old fermentation operating in a 100 L internal loop airliftbioreactor with Neurospora crassa biomass and 30 g/l sucrose, 3 g/l Na3citrate, 2 g/l KH2PO4, 2 g/l NH4NO3, 0.2 g/l MgSO4, 1 g/l CaSO4, 0.005g/l Zn SO4, 0.001 g/l Fe(NH4)2(SO4)2, 0.00025 g/l CuSO4, 0.0001 g/lMnSO4, 0.0025 g/l biotin was injected, via peristaltic pumping, with 10g/l of powdered dried oats that had been steam sterilized in four, 1liter glass bottles with DI water adjusted to the one liter mark on eachbottle. The fermentation was continued with the previous conditions fora remaining 8 hours. The material was harvested and processed withtypical methods.

Example 8

The dry TCP described herein was fully characterized in its nutritionalprofile and texture. The TCP was cultivated in a 100 L internal loopairlift bioreactor for 3 days at 31 C with 0.7 VVM of compressed air and40 g/l sucrose, 3 g/L sodium citrate, 3.5 g/L ammonium phosphate, 1.3g/l potassium nitrate, 0.1 g/l magnesium sulfate, 0.05 g/l calciumchloride and minor amounts of copper, iron, manganese, and zinc. Thematerial was harvested in slurry form, de-watered down to 75% WC using avibratory screen and screw press, compacted into a dense block, shreddedwith a spindle and tine shredding device, sized with a 5 mm mesh, anddehydrated with a forced air conveyor dryer. The material was baked at83 C in a convection oven for 15 minutes for a final pasteurization. Thematerial with an ambient hydration of 4.3% water content (WC) wasanalyzed for a full nutritional profile via triplicate sampling. Thefollowing are averages. Using AOAC method 991.43, fiber content wasdetermined to be 18.14%. Using AOAC method 990.03 total protein wasdetermined to be 53%. Using AOAC method 945.44 total fat was determinedto be 9.02%. Using AOAC methods for individual amino acids the followingconcentrations were determined: Methionine.53%, Cystine 0.47%, Lysine2.49, Phenylalanine 1.35%, Leucine 3.02%, Isoleucine 1.53%, Threonine1.79%, Valine, 2.84%, Histidine 1.61%, Arginine 3.07%, Glycine 1.52%,Aspartic acid 3.91%, Serine 1.74%, Glutamic acid 5.78%, Proline 1.44%,Hydroxyproline 0.04%, Alanine 2.51%, Tyrosine 2.17%, and Tryptophan0.42%, Taurine.01%. AOAC method 2012.13 was used to determine the fattyacid profile which was determined to be (relative %): Total omega 3content 3.3%, total omega 6 content 35.9%, total omega 9 content 38.3%,total saturated 17.8%, total monounsaturated 42.7%, and totalpolyunsaturated 39.3%. Using AOAC method 990.12 the aerobic plate countwas determined to be <10,000 cfu/g. Using AOAC method 997.02 the moldand yeast count was determined to be <200 CFU/g. Using AOAC method991.14 the E. coli levels were determined to be <10/g. Using AOAC method2003.09 the Salmonella spp. Was determined to be negative in 375 gramsof TCP.

The aforementioned TCP was then characterized in its texture andmacro-scale characteristics after hydration. Color was visuallydetermined to be an off white/tan. Particle size was determined to be anaverage of 5 mm on the x, y, and z planes. Water absorption capacity(WAC) was determined to be 4624.77 g/kg. The chewiness, cohesiveness,springiness, as well as transversal and longitudinal cutting strengthwere analyzed to determine “texture”. The chewiness was determined to be3.43 kg. The cohesiveness was determined to be 45%. The springiness wasdetermined to be 62%. Transversal cutting strength was determined to be12,408 kg/m⁻². Longitudinal cutting strength was determined to be 11,316kg/m⁻². These numbers were compared to results from beef, chicken, andpork.

Example 9: Blended Beef/TCP Patties

60 g immobilized TCP of Rhizopus oryzae hydrated at 72% water content/60g ground chuck beef

Fungal biomass was hydrated and added to the ground beef after theinitial course grind. The components were then mixed/blended into thedesired consistency for burger patties.

The ground beef/fungi blend was packed into a circular mold with adiameter of 120 mm.

The patty was cooked in an oiled pan on medium heat until internaltemperature reached 73 C.

Example 10: Blended Chicken/TCP Breaded Nuggets

70 g immobilized TCP of Aspergillus oryzae hydrated at 65% watercontent/30 g ground white meat chicken

Fungal TCP was hydrated and added to shredded chicken breast meat. Themixture was tossed and ground through plates with ⅛″ holes.

Meat blend was scooped into 10 gram nuggets. The nuggets were compressedinto a breading consisting of 80% whole wheat flower, 18.3% breadcrumbs, 0.6% salt, 0.2% paprika, 0.2% basil, 0.2% cayenne pepper, 0.2%garlic powder, 0.3% ground black pepper by dry weight. Compression wasapplied to both sides of the nugget, simultaneously covering the entiremeat blend and flattening the nugget on two sides.

Nuggets were fried in ¼″ depth peanut oil until breading was crispy.

Example 11: Blended Pork/TCP Sausages

130 g immobilized TCP of Neurospora intermedia hydrated at 68% watercontent/80 g ground pork/30 g pork fat

TCP was hydrated and added to ground pork and fat mixture. Contents weremixed with 0.2 g salt, 0.05 g powdered sage, 0.1 g ground black pepper,0.2 g Italian herbs, 0.1 g paprika. Mixture was mixed together using amechanical mixer for 4 minutes. Mixture was put through a grinder with⅛″ holes.

Ground and blended material was injected into a cleaned and preparednatural hog sausage casing and tied off to close. The sausage wastwisted and cut to produce two sausage links.

Links were pan seared in a pan on medium heat until center temperaturereached 71 C.

Example 12: Dry Chicken Extender

80 g of immobilized Neurospora sitophila TCP at 6% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded, sized into 5mm particles, and consecutively dried down to 6% water content. This TCPwas then baked in an oven at 85° C. for 15 minutes for a finalpasteurization. 80 g of the TCP was mixed and coated with 10 g saffloweroil, 5 g algae, 5 g natural chicken flavoring.

The composition had particle sizes of 3-10 mm. The plant basedingredients adhered evenly to the surfaces of the dehydrated myceliumparticles. The mixture had a tan color and tasted like dry chicken.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground chicken in a mixer until texturally homogenous(roughly 30 seconds). The blended chicken was formed into patties andcooked in the same way as chicken patties.

Example 13: Dry Pork Extender

72 g of immobilized Neurospora intermedia TCP at 5.5% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded, sized into 5mm particles, and consecutively dried down to 6% water content. This TCPwas then baked in an oven at 85° C. for 15 minutes for a finalpasteurization. 72 g of the TCP was mixed and coated with 10 g saffloweroil, 2 g yeast extract, 6 g bamboo fiber, 10 g natural pork flavoring.

The composition had particle sizes of 3-10 mm. The plant-basedingredients adhered evenly to the surfaces of the dehydrated myceliumparticles. The mixture had a tan color and tasted like dry pork.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground pork in a mixer until texturally homogenous(roughly 30 seconds). The blended pork was formed into sausage links andcooked in the same way as pure pork sausages.

Example 14: Dry Beef Extender

64 g of immobilized Neurospora crassa TCP at 6% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded, sized into 5mm particles, and consecutively dried down to 6% water content. Thismaterial was then baked in an oven at 85° C. for 15 minutes for a finalpasteurization. 64 g of the material was mixed and coated with 10 gsafflower oil, 2 g yeast extract, 5 g psyllium husk, 10 g beet pulppowder, 10 g natural beef flavoring.

The composition had particle sizes of 3-10 mm. The plant-basedingredients adhered evenly to the surfaces of the TCP particles. Themixture had a tan color and tasted like dry beef.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground beef in a mixer until texturally homogenous(roughly 30 seconds). The blended beef was formed into burger pattiesand cooked in the same way as pure beef burgers.

Example 15: Dry Chicken Extender

80 g of immobilized Neurospora sitiphila TCP at 6% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded andconsecutively dried down to 6% water content. This material was thenbaked in an oven at 85° C. for 15 minutes for a final pasteurization. 80g of the TCP was mixed and coated with 10 g safflower oil, 5 g algae, 5g natural chicken flavoring.

The composition had particle sizes of 3-10 mm. The plant basedingredients adhered evenly to the surfaces of the TCP particles. Themixture had a tan color and tasted like dry chicken.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground chicken in a mixer until texturally homogenous(roughly 30 seconds). The blended chicken was formed into patties andcooked in the same way as chicken patties.

Example 16: Dry Pork Extender

72 g of immobilized Neurospora intermedia at 5.5% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded andconsecutively dried down to 6% water content. This material was thenbaked in an oven at 85° C. for 15 minutes for a final pasteurization. 72g of the TCP was mixed and coated with 10 g safflower oil, 2 g yeastextract, 6 g bamboo fiber, 10 g natural pork flavoring.

The composition had particle sizes of 3-10 mm. The plant basedingredients adhered evenly to the surfaces of the TCP particles. Themixture had a tan color and tasted like dry pork.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground pork in a mixer until texturally homogenous(roughly 30 seconds). The blended pork was formed into sausage links andcooked in the same way as pure pork sausages.

Example 17: Dry Beef Extender

64 g of immobilized Neurospora crassa at 6% water content

Raw mycelium was harvested and de-watered down to 70% water contentusing a screen and screw press. This material was shredded andconsecutively dried down to 6% water content. This material was thenbaked in an oven at 85° C. for 15 minutes for a final pasteurization. 64g of the material was mixed and coated with 10 g safflower oil, 2 gyeast extract, 5 g psyllium husk, 10 g beet pulp powder, and 10 gnatural beef flavoring.

The composition had particle sizes of 3-10 mm. The plant-basedingredients adhered evenly to the surfaces of the TCP particles. Themixture had a tan color and tasted like dry beef.

The composition was hydrated at a 1/1.5 ratio of dry material to waterby weight. This hydrated material was mixed at a 30/70% ratio ofcomposition to ground beef in a mixer until texturally homogenous(roughly 30 seconds). The blended beef was formed into burger pattiesand cooked in the same way as pure beef burgers.

Example 18: Focus Group On TCP in Blended Meat Application

The dry shelf-stable ingredient of the present invention with a D-50 of4 mm at 75% w/w was coated in safflower oil at 10% w/w and blended with“brown chicken” natural flavor (Fontana Flavors) at 10% w/w as well aspea fiber at 5% w/w. The coated particles were hydrated at a 1/2 ratioof particles/water w/w (200% hydration) and left to soak for 25 minutes.The hydrated TCP was mixed with ground chicken meat at a 50/50 ratiow/w. The blend was formed into 16 g nuggets, then breaded in a standardnugget breading consisting of rice flour, wheat flour, cornmeal, andspices.

Pure ground chicken was formed into 16 g nuggets and breaded with theexact same breading formula. Both the pure chicken and the blendednuggets were fried until they floated in the oil and had a golden crispybreading. The products were kept separate, given the respective ID's of1776 for fully chicken and 1865 for the blended nugget.

20 random testers were selected from a bank of participants unfamiliarto the project. They were requested to taste the two samples and torecord their preference using three options; “X preferred” or “cannottell the difference”. The testers were served each sample with a shortbreak in between. Their results were recorded.

12 said they could tell no difference. 6 preferred the blended nugget,and 2 preferred the fully meat nugget. (60% said no difference, 30%preferred the blended nugget, 10% preferred the full chicken nugget).

Example 19: Treatment Efficiency with Potato Processing Wastewater

Cultures of Neurospora sitophila were cultured in two 200 ml baffledshaker flasks with house media for 30 hours at 100 RPM and 31 C. Mediawas prepared in agitated 100 L pressure vessels and steam sterilized toprepare 16 L of media. The media consisted of potato processing wastewater generated from the blanching of potatoes for french fryproduction. The water contained a high COD of 37,304 mg/L COD, 2.34%TSS, 1397 mg/L TN, 3.5% sugars, as well as phosphate and sulphate. Themedia was supplemented with 1 g/L yeast extract, 0.5 g/L ammoniumnitrate, and 10 ml of vegetable oil based antifoam. The media was steamsterilized and injected into the steam sterilized 17 L reactor. The seedcultures (400 ml total) were added to the reactor. The fermentation wasmaintained at 31 C, a pH of 4.5 and 0.8 vvm of compressed air for threedays (72 hours). The material was harvested and processed as typicalwith the methods of the present invention.

The supernatant was analyzed. Remaining COD was 1283 mg/L, TSS was0.01%, TN was 188 mg/L and sugars was 0%. The treatment was highlyeffective in reducing COD as well as TSS and TN. In addition to creatinga quality TCP from the process, the treatment capacity of the platformproves viable as a treatment to the potato processing waste.

What is claimed is:
 1. A shelf-stable protein food ingredientcomprising: filamentous fungal particles from the genus Neurospora witha mean particle size between about 5 mm and about 20 mm or between about5 mm and about 50 mm, said filamentous fungal particles consistingessentially of: a) cultured filamentous fungal biomass from the genusNeurospora in an amount at least about 94-96% w/w; and b) water in anamount of about 4% to about 6% w/w.
 2. The shelf-stable protein foodingredient of claim 1 with a mean particle size of about 8 mm.
 3. Theshelf-stable protein food ingredient of claim 1, wherein the fungalbiomass is from the species, Neurospora intermedia, Neurosporasitophila, Neurospora crassa, or a combination of Neurospora intermedia,Neurospora crassa, and Neurospora sitophila.
 4. The shelf-stable proteinfood ingredient of claim 1 further comprising fat in an amount of 1-80%.5. The shelf-stable protein food ingredient of claim 1, furthercomprising albumin, pectin, silicone dioxide, zinc gluconate, vitaminB12, maltodextrin, niacin, sodium ascorbate, pyridoxine hydrochloride,tetrasodium pyrophosphate, calcium carbonate, sodium alginate, alginate,trisodium phosphate, calcium acetate, methylcellulose, cellulose, bamboocellulose, annatto, acetic acid, sodium nitrite, sodium benzoate, soylecithin, or any combination thereof.
 6. A food ingredient compositioncomprising one or more plant ingredients and the shelf-stable proteinfood ingredient of claim
 1. 7. A food product comprising theshelf-stable protein food ingredient of claim 1, further comprising: (a)one or more meat ingredients; (b) one or more plant ingredients; or (c)both one or more meat ingredients and one or more plant ingredients. 8.The food product of claim 7, wherein the shelf-stable protein foodingredient with further comprises water and the one or more plantingredients.
 9. The food product of claim 7, wherein the shelf-stableprotein food ingredient further comprises oil and the one or more plantingredients.
 10. The food product of claim 7, wherein the shelf-stableprotein food ingredient further comprises water and the one or more meatingredients.
 11. The food product of claim 7, wherein the shelf-stableprotein food ingredient further comprises the one or more meatingredients.
 12. The food product of claim 7, wherein the one or moremeat ingredients is selected from beef, pork, chicken, turkey, fish,lamb, crab, lobster, venison, bison, and combinations thereof.
 13. Amethod for producing a shelf-stable food ingredient comprising the stepsof: a) culturing a filamentous fungi from the genus Neurospora in aliquid growth medium to produce a filamentous fungal biomass slurrycomprising about 0.5-8% biomass; wherein the liquid growth mediumcomprises 10-30 g/l sucrose or glucose, 2.0-5.0 g/l KH₂PO₄, 0.5-2.0 g/lNH₄NO₃, 0.2 g/l MgSO₄, 1 g/l CaSO₄, 0.005 g/l Zn SO₄, 0.001 g/lFe(NH₄)²(SO₄)², 0.00025 g/l CuSO₄, 0.0001 g/l MnSO₄, and 0.0025 g/lbiotin; b) harvesting the filamentous fungal biomass and dewatering thefilamentous fungal biomass to produce a harvested filamentous fungalbiomass comprising about 60-85% water and about 15-40% filamentousfungal biomass; c) pressing the harvested filamentous fungal biomass toproduce a filamentous fungal biomass slab; d) shredding and sizing thefilamentous fungal biomass slab to form filamentous particles with amean particle size between about 5 mm and about 20 mm or between about 5mm and about 50 mm; and e) drying the particles at about 50° C. to about85° C. to form the shelf-stable food ingredient.
 14. The method of claim13, wherein the filamentous fungal biomass slurry is grown by aerobicfermentation.
 15. The method of claim 13, wherein the liquid growthmedium further comprises starches, fatty acids, sugars, minerals, traceelements, vitamins, extracts, or combinations thereof.
 16. The method ofclaim 13, wherein one or more of the liquid growth medium components arederived from plant ingredients, or potato processing wastewater, or cornstillage byproducts.
 17. The method of claim 13, wherein one or more ofthe liquid growth medium components are derived from fruit pulp, grainprocessing, distillation byproducts, brewing byproducts, corn stillage,potato processing waste, potato blanche water, rice processing waste,wheat straw, dairy whey, coffee processing waste, soda manufacturingwaste, molasses, sugarcane bagasse, vinasse, cassava processing waste,or any combination thereof.
 18. The method of claim 13, wherein thefilamentous fungal biomass slab is shredded, then sized by continuoussieving using 2 mm and 12 mm sieves.
 19. The shelf-stable protein foodingredient of claim 1, wherein the filamentous fungal particles have achewiness ranging between about 0.5 kg and about 15 kg; a cohesivenessranging between about 30% and about 80%; and a springiness rangingbetween about 20% and about 85%, when hydrated at a 1/1.75 ratio of drymaterial to water by weight.
 20. The shelf-stable protein foodingredient of claim 1, wherein the filamentous fungal particles have amean particle size between about 4 mm and about 10 mm.
 21. Theshelf-stable protein food ingredient of claim 1, wherein the filamentousfungal particles have a mean particle size between about 5 mm and about10 mm.
 22. The shelf-stable protein food ingredient of claim 1, whereinthe filamentous fungal particles have a mean particle size between about6 mm and about 50 mm.
 23. The shelf-stable protein food ingredient ofclaim 1, wherein the filamentous fungal particles have a mean particlesize between about 6 mm and about 20 mm.
 24. The shelf-stable proteinfood ingredient of claim 1, wherein the filamentous fungal particleshave a mean particle size between about 7 mm and about 20 mm.
 25. Theshelf-stable protein food ingredient of claim 1, wherein the filamentousfungal particles have a mean particle size between about 7 mm and about15 mm.
 26. The shelf-stable protein food ingredient of claim 1, whereinthe filamentous fungal particles have a mean particle size between about7 mm and about 12 mm.
 27. The shelf-stable protein food ingredient ofclaim 1, wherein the filamentous fungal particles comprise about 30-70%protein.
 28. The shelf-stable protein food ingredient of claim 1,wherein the filamentous fungal particles have a mean particle sizebetween about 5 mm and about 20 mm or between about 5 mm and about 50mm, and a diameter of about 6 mm, wherein about 50% of the mass of theshelf-stable protein food ingredient is in particles less than about 6mm in diameter.
 29. The method of claim 13, wherein the filamentousparticles have a mean particle size between about 5 mm and about 20 mmor between about 5 mm and about 50 mm, and a diameter of about 6 mm,wherein about 50% of the mass of the shelf-stable protein foodingredient is in particles less than about 6 mm in diameter.
 30. Themethod of claim 13, wherein the liquid growth medium further comprises 3g/l Na₃ citrate.
 31. The method of claim 13, wherein the dried particlescomprise water in about 4% to 6% w/w.
 32. The shelf-stable protein foodingredient of claim 1, wherein the shelf-stable protein food ingredienthas a water holding capacity between about 2,000 g/kg to about 7,000g/kg.
 33. The shelf-stable protein food ingredient of claim 1, whereinthe shelf-stable protein food ingredient is hydrated at about a 1/1.5 toabout a 1/2 ratio of dry material to water by weight.